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Preparation method of immobilized cis-epoxysuccinic acid hydrolase gene engineering bacterial cells and D(-)-dihydroxysuccinic acid or salt thereof

A technology of epoxy succinic acid and genetically engineered bacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, fixed on/in organic carriers, etc., can solve the problems of high product impurities, high cost, and large pollution, etc. problems, to achieve high cell utilization, high enzyme activity, optical purity and high yield

Active Publication Date: 2011-04-13
HANGZHOU BIOKING BIOCHEM ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Genetically engineered bacterial cells with high cis-epoxysuccinate hydrolase activity can only be used once, resulting in high cost and large pollution
[0006] (2) The cis-epoxysuccinate hydrolase activity of genetically engineered bacterial cells is low, generally 3278U / g
Since cis-epoxysuccinate hydrolase is an intracellular enzyme, the opportunity to contact with the substrate is limited by the permeability of the cell membrane, resulting in a decrease in its catalytic activity
[0007] (3) There are many impurities in the product
But there is no report about the production of D(-)-tartaric acid by immobilized cells at home and abroad

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] According to the method disclosed in the Chinese invention patent application CN101481681A, the cis-epoxysuccinate hydrolase genetically engineered bacterial cell pET22b-ESH-E.coli BL21(DE3) was prepared. 1 g of the episomal engineered bacterial cell pET22b-ESH-E.coliBL21(DE3) harvested by high-speed centrifugation (5000×g, 10min) at 4°C (the cis Formula epoxy succinate hydrolase activity is 3278U / g) was added to 10ml 30g / L κ-carrageenan solution, mixed evenly at 42°C, cooled at 4°C, solidified with 0.3mol / L chlorine Potassium chloride solution was fixed at 4°C for 10 h. Take part of the gel and cut the gel into 3*3*3mm 3 The small pieces were washed successively with distilled water and normal saline respectively. The results showed that the cis-epoxysuccinate hydrolase activity of the immobilized genetically engineered bacteria pET22b-ESH-E.coliBL21(DE3) was 29047U / g, and the mechanical strength reached 1107g / cm 2 .

Embodiment 2

[0043] Immobilized genetically engineered bacterial cells were prepared according to the method of Example 1, wherein the amount of genetically engineered bacterial cells added was 1 g, the concentration of κ-carrageenan was 20 g / L, and other conditions were unchanged. The results showed that the cis-epoxysuccinate hydrolase activity of the prepared immobilized genetically engineered bacteria was 22935U / g, and the mechanical strength reached 1664g / cm 2 .

Embodiment 3

[0045] Immobilized genetically engineered bacterial cells were prepared according to the method of Example 1, wherein the amount of genetically engineered bacterial cells added was 1 g, the concentration of κ-carrageenan was 40 g / L, and other conditions were unchanged. The results showed that the cis-epoxysuccinate hydrolase activity of the prepared immobilized genetically engineered bacteria was 31777U / g, and the mechanical strength reached 1019g / cm 2 .

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Abstract

The invention discloses a preparation method of immobilized cis-epoxysuccinic acid hydrolase gene engineering bacterial cells and D(-)-dihydroxysuccinic acid or salt thereof, comprising the following steps: adding immobilized cis-epoxysuccinic acid hydrolase gene engineering bacterial cells in k-kara peptizing liquid to sufficiently and evenly mix; cooling the mixed solution to sufficiently solidify and fixing in a potassium chloride solution; taking out gel to be cut into small blocks; and sequentially washing the small blocks with distilled water and physiological salt solution. The immobilized gene engineering bacterial cells are added into the cis-epoxysuccinic acid or the salt solution thereof to carry out enzymic reaction so as to produce the D(-)-dihydroxysuccinic acid or the salt thereof. The activity of the cis-epoxysuccinic acid hydrolase of the immobilized cis-epoxysuccinic acid hydrolase gene engineering bacterial cells pET-ESH-E.coli is more than 4 times of free cis-epoxysuccinic acid hydrolase gene engineering bacterial cells so that the cis-epoxysuccinic acid hydrolase of the immobilized cis-epoxysuccinic acid hydrolase gene engineering bacterial cells pET-ESH-E.coli can be recycled, and the optical purity and the yield of the D(-)-dihydroxysuccinic acid product are improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing immobilized cis-epoxysuccinate hydrolase genetically engineered bacterial cells, and using immobilized cis-epoxysuccinate hydrolase genetically engineered bacterial cells to prepare D(-)- method of tartaric acid or its salts. Background technique [0002] D(-)-tartaric acid, also known as (2S,3S)-2,3-dihydroxybutane-1,4-dicarboxylic acid, is the isomer of natural L(+)-tartaric acid, which is very common in nature. It rarely exists, and it is mainly used as a chiral source and resolution agent for chiral synthesis in the pharmaceutical industry. At present, the demand for D(-)-tartaric acid is increasing year by year, and there is an urgent need to increase production to meet domestic and foreign demands. [0003] The current methods for producing D(-)-tartaric acid include chemical resolution, biological resolution, and biotransformation. The chemical resoluti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/10C12P7/46C12R1/19
Inventor 张建国谢志鹏潘海峰鲍文娜
Owner HANGZHOU BIOKING BIOCHEM ENG